Research on aspartate transcarbamylase (ATCase) and other enzymes is aimed at determining the relationship between tertiary and quaternary structures and the effect of aggregation on the folding of polypeptide chains. Native and chemically modified proteins are being studied by techniques such as sedimentation equilibrium and velocity, circular dichroism, spectrophotometry, fluorescence, and stopped-flow kinetics. Results on subunits will be correlated with those for intact molecules. With ATCase, for example, the appropriately weighted sum of the circular dichroic spectra of the separate subunits does not equal that obtained for the intact enzyme. Absorption spectra for the enzyme containing regulatory subunits bearing a covalently attached chromophore indicate that there are local conformational changes when the intact enzyme is assembled from the individual subunits. Other probes will be used for an analysis of these conformational changes. The assembly of the 12 polypeptide chains of ATCase from two catalytic trimers and three regulatory dimers is being studied in order to search for partially assembled "stable" intermediates and to clarify the sequence of steps in the formation of the native enzyme. The strengths of the inter-subunit bonding domains are being assessed from association-dissociation equilibria analyzed by a difference sedimentation equilibrium technique using an improved photoelectric scanner. Chemical variants and mutants from E. coli as substrates (homotropic effect) and their response to various inhibitors and activators (heterotropic effect). Our goals are to determine whether these effects are inextricably linked and to elucidate the nature of the cooperative unit in this regulatory enzyme. Analogous investigations on native and chemically modified aldolase which shows no cooperativity are aimed at exploring the concepts of quaternary constraint to see if single polypeptide chains which cannot associate with one another can be "rescued" by other chains which are inactive but can aggregate specifically. Our goal is to determine whether enzyme activity can be generated in this process.